Individuals suffering from diabetes mellitus have an abnormally high blood sugar level because the pancreas does not secrete sufficient amounts of the hormone insulin into the bloodstream to regulate carbohydrate metabolism. If an abnormally high blood sugar level, i.e. a hyperglycemic condition, is allowed to continue for prolonged periods, the individual will suffer from the chronic complications of diabetes, including retinopathy, nephropathy, neuropathy and artherosclerosis.
Accordingly, it is important that a hyperglycemic condition, or excess sugar in the blood, be controlled in order to prevent, or ameliorate, the chronic complications of diabetes. Therefore, in order to prevent the chronic complications of diabetes, the blood glucose level of a diabetic individual is maintained as closely as possible to the blood glucose level found in healthy individuals. Usually, blood glucose levels are maintained by periodically injecting insulin into the individual in order to provide the hormone necessary for glucose metabolism.
As a result, reliable test methods are needed in order for the individual, or a physician, to determine blood sugar levels such that a physiologic abnormality can be properly treated, or diagnosed, and such that a proper treatment schedule is instituted and maintained. Unfortunately, many of the prior art methods of determining the amount of glucose in the blood are unsatisfactory either because the method requires patient cooperation, such as fractional urine collections by the individual at home, or because the method is cumbersome and unreliable, such as 24 hour urinary glucose excretion. In addition, blood glucose levels fluctuate considerably throughout the day because of the influences of diet, physical activity, and medical treatment. Therefore, random blood glucose assays are an inadequate and/or misleading indicia of blood sugar control.
Consequently, in attempting to control an established case of diabetes mellitus, an accurate assay of the actual, and not the apparent, long-term blood glucose level is necessary. An early prior art method measured the amount of glycosylated hemoglobin in the blood as an indicator of blood sugar, or glucose, concentration. This assay has proved useful in monitoring the control of diabetes mellitus, but the assay is expensive and assay results are relatively nonreproducable. Therefore, the need for a more reliable assay for glucose concentration has led to the present investigation, wherein a fast and accurate fructosamine assay can be correlated to the glucose concentration of the liquid test sample.
Fructosamines are a product of the interaction of serum glucose with serum proteins over a period of days to weeks. An assay for fructosamines overcomes some of the disadvantages of the assay for glycosylated hemoglobin because fructosamines are stable, and fructosamine concentration does not change with short term fluctuations in blood glucose levels. As will be demonstrated more fully hereinafter, the fructosamine assay of the present invention also is economical, accurate, and reproducible, and can be performed on one or two drops of test sample.
It is known that proteins, such as those present in blood serum, react with glucose either in vivo or in vitro, under non-enzymatic conditions, to produce glycated proteins, also known as fructosamines or ketoamines. In addition, the fructosamine concentration of a test sample can be correlated to diabetic glycemic control, especially relating to the 3 to 6 weeks prior to testing. Furthermore, the amount of fructosamine present in the blood also has been found to correlate with fasting plasma glucose and hemoglobin A.sub.Ic (HbA.sub.Ic) in individuals suffering from diabetes. In addition, both the time of taking the liquid test sample and the relation of the test sample to diet are unimportant.
The glycosylation of proteins to form fructosamines is a non-enzymatic reaction. The extent of the reaction is directly dependent upon the glucose concentration of the blood. Consequently, diabetics usually have an elevated fructosamine concentration in the blood compared to a healthy individual. Therefore, the concentration of glycated serum proteins or glycated hemoglobin has been used as an indicator of a glycemic condition. In particular, an assay for fructosamines provides a method of diagnosing for diabetes mellitus and of monitoring diabetic control measures because fructosamine concentration reflects an average of serum glucose levels over a period of time.
It is known that blood glucose reacts continuously with proteins. The glucose binds to an amino group of a protein to form a Schiff base, i.e., an aldimine, that undergoes a molecular rearrangement to form a stable ketoamine In the art, such ketoamines are generically termed "fructosamines". Therefore, in accordance with the method of the present invention, blood sugar concentrations are determined indirectly by assaying for fructosamines.
The formation of fructosamines is demonstrated in the following chemistry scheme The blood proteins are glycated in vivo by a non-enzymatic reaction between glucose and available amino groups of blood proteins. ##STR1## The degree of protein glycosylation is directly proportional to blood glucose concentration. In general, the fructosamine concentration in the blood reflects an average glucose concentration in the blood over a period of time. Therefore, diabetic individuals have a higher fructosamine concentration in the blood than unaffected individuals. Then, under alkaline conditions, the fructosamines that form in the blood are converted to eneaminols. The eneaminol form of fructosamine is a chemically active reducing substance that reacts with a suitable indicator dye to give a color transition that can be measured and compared with a standard to give an indication of the glucose concentration in blood samples This correlation to glucose concentration is possible even though the glucose remains interacted with or associated with the protein in the blood and has undergone a molecular rearrangement to form a fructosamine. ##STR2##
Fructosamine concentration in the blood is an indicator of a diabetic condition, and is an indicator of diabetic control measures that are dependent on the patient's diet, activity and/or treatment. The major clinical use of a fructosamine assay is the diagnosis of diabetes, the regulation of treatment for known diabetics and the assessment of new diabetes treatments. A rapid and accurate fructosamine assay also can be used to screen populations with an increased risk of developing diabetes, such as pregnant women, the obese, and the ethnic groups with a known predisposition to diabetes. Therefore, if a rapid and accurate fructosamine assay of blood samples were available, the assay could assess glycemic control, therapeutic regimen and patient compliance over extended time periods in order to avoid the complications of diabetes associated with hyperglycemia. Accordingly, the availability of a trustworthy fructosamine plasma or serum diagnostic assay would compliment the battery of assays, such as plasma glucose and HbA.sub.Ic, presently performed by physicians in the diagnosis of diabetes and in the care and treatment of diabetics.
Baker, in U.S. Pat. Nos. 4,642,295 and 4,645,742, and in European patent application No. 82307051.1, discloses a method of determining fructosamine levels in blood samples based upon the above-described chemical scheme The method disclosed by Baker is a wet phase assay, wherein the blood serum must be separated from the highly-colored red blood cells. After separation of the serum from the blood cells, the serum sample is buffered to a pH of between 10 and 11 and a coloring agent, nitroblue tetrazolium, is added. The resulting mixture is allowed to stand for 10 to 15 minutes, then any color change resulting from a reaction between the nitroblue tetrazolium and the eneaminol form of the frustosamine is measured. In order for the assay to proceed in a relatively rapid fashion, the buffered serum sample containing the nitroblue tetrazolium is maintained at 37.degree. C., or 98.6.degree. F., throughout the assay. However, the method of Baker suffers from several practical limitations such as the inconvenience of a wet phase assay; the need for a relatively large blood sample volume of about 2 ml; the elevated temperature required to effect the reaction; the length of time to complete the assay; and the need to separate blood serum from blood cells. These drawbacks make laboratory assays for fructosamines time-consuming and burdensome, and such practical limitations are especially disadvantageous for an individual desiring to conduct a fructosamine assay at home.
German Patent No. DE 36 20 817 describes a wet phase chromogenic assay for fructosamines that first eliminates the interfering components in the serum by treating the serum with a lipase and either an oxidase, a peroxidase or a catalase, at a neutral pH. This method eliminates any non-specific reducing components and turbidity-forming components from the serum. Then, the serum is assayed for fructosamine concentration at a pH of 10 to 11. In contrast, the composition and method of the present invention is a dry phase assay for fructosamines that is essentially unaffected by the non-specific reducing components. Furthermore, the present invention does not require the time-consuming manipulative steps of adjusting the pH to a value of 10 to 11, then adding the indicator dye.
Japanese Kokai Patent No. Sho 60 (1985)-227171 discloses a method of assaying for blood sugar by determining the concentration of glucose combined with hemoglobin. The Japanese patent discloses a wet assay method wherein a hemolyzed blood sample, preferably plasma-free, is mixed with nitroblue tetrazolium in a composition that first converts the hemoglobin from a tetramer to a monomer. Then, the glucose bound to the hemoglobin (as fructosamine) is determined spectrophotometrically. The Japanese patent does not teach or suggest using a dry phase test strip to assay blood or serum for glucose concentration by determining the amount of glucose bound to a protein in the fructosamine form. Furthermore, like the other prior art fructosamine assays, the Japanese method is a wet assay method requiring several manipulative steps, such as preferably using separated and washed blood cells to avoid assay interference attributed to fructosamines arising from glucose bound to proteins.
Therefore, the need exists for a rapid, accurate fructosamine assay of blood samples. The fructosamine assay should be an easy-to-perform assay, either at home or in a medical office, in order to rapidly determine if a glycemic condition exists and to rapidly determine the efficacy of the prescribed medical treatment for diabetes. Accordingly, investigators have attempted, and failed until the present invention, to provide a fructosamine assay in the dry phase test strip format. At present, no commercial fructosamine test strips are available, and no fructosamine test strips are known to be disclosed in prior art patents or publications.
Consequently, in order to determine if an individual is hyperglycemic, i.e., has an excess amount of sugar in the blood, or is hypoglycemic, i.e., has a lack of sugar in the blood, and in order to monitor the course of a medical treatment to determine the effectiveness of that treatment, a simple, accurate and inexpensive assay that correlates to the amount of sugar in the blood is needed. Although semiquantitive, colorimetric reagent test strip assays are available for the determination of sugar content in a test sample, until the method and device of the present invention no dry phase colorimetric test strips were available to indirectly assay for blood sugar content by measuring fructosamine content.
In general, a colorimetric reagent test strip utilizes the ability of the analyte of interest to interact with certain reagents present in the test pad of the test strip and to ultimately alter the color of an indicator present in the test pad. Some colorimetric test strips used in blood assays have a single test area consisting of a small square pad of a carrier matrix impregnated with the reagents necessary to interact with the analyte of interest to produce a detectable response. Other colorimetric test strips are multideterminant reagent strips that include one test area for the assay of a particular analyte, and further include several additional test areas on the same strip to permit the simultaneous assay of other blood constituents. For both types of colorimetric test strips, the assay for a particular analyte is performed by simply dipping the colorimetric test strip into a fresh whole blood, or blood serum, sample, then comparing the resulting color of the test area of the test strip to a standardized color chart provided on the colorimetric test strip bottle.
However, until the test device of the present invention, no dry phase test strips were available to allow an accurate determination of fructosamine content in blood. Therefore, it would be extremely advantageous to have a simple, accurate and trustworthy method of assaying blood or blood serum for fructosamine content that allows visual differentiation of fructosamine levels in the range of from about 0.5 mMol/L to about 6 mMol/L, and especially in the range of from about 1 mMol/L to about 4 mMol/L. The fructosamine concentration in the blood then can be correlated to the amount of glucose in the blood to provide an indirect blood sugar measurement. By providing an accurate method of determining the fructosamine concentration in an easy to use form, like a dip-and-read test strip, the blood assay can be performed by laboratory personnel to afford immediate test results such that correct medical treatment can be commenced immediately. In addition, the test strip method can be performed by the patient at home to more precisely monitor fructosamine levels, and therefore sugar levels, in the blood as a measure of the success of the medical treatment the patient is undergoing.
As will be described more fully hereinafter, the method of the present invention allows the fast, accurate and trustworthy fructosamine assay of blood by utilizing a test strip that includes an indicator reagent composition. The indicator reagent composition of the present invention provides sufficient visual color resolution between different fructosamine concentrations, and therefore the assay has sufficient sensitivity to allow fructosamine concentrations to be accurately determined. All prior art assay techniques for fructosamines involve wet assay methods that are time consuming and are not amenable for use by the patient at home.
Therefore, incorporating a highly sensitive indicator reagent composition into a suitable carrier matrix provides a test pad for use in a dry phase test strip of the present invention that allows the rapid, room temperature determination of fructosamine levels in blood or serum. The detection and measurement of fructosamines in blood can be correlated to the amount of sugar in the blood such that a correct diagnosis for glycemia can be made, and such that correct medical treatment can be implemented, monitored and maintained. In addition, because the fructosamine assay is in a dip-and-read format, the assay provides for the easy and economical, qualitative or quantitative determination of fructosamines in blood or other liquid test samples.
Any colorimetric method of assaying for fructosamine in blood or other liquid test samples must yield accurate, trustworthy and reproducible results by utilizing a reagent composition that undergoes a color transition as a result of an interaction with fructosamines, and not as a result of a competing chemical or physical interaction, such as a pH change or a preferential interaction with a test sample component other than fructosamines. Additionally, the method and composition utilized in the assay for fructosamines should not adversely affect or interfere with the other test reagent pads that are present on multiple test pad strips.
Prior to the present invention, no known method of assaying blood or other liquid test samples for fructosamines included an indicator reagent composition incorporated into a suitable carrier matrix to provide sufficient color resolution of the assay and sufficient sensitivity of the assay to fructosamine concentrations, such that an accurate and trustworthy fructosamine assay is available for fructosamine concentrations ranging from about 0.5 mMol/L to about 6 mMol/L, and especially from about 1 mMol/L to about 4 mMol/L. Furthermore, until the method of the present invention, no dry phase test strip procedures were available to assay for the fructosamine concentration in blood or other liquid test samples. However, surprisingly and unexpectedly, the method of the present invention permits the dry phase test strip assay of blood and other liquid test samples for fructosamines, in order to diagnose a glycemic condition and to monitor treatments for glycemic conditions.
In contrast to the prior art assay procedures, the method of the present invention provides sufficient sensitivity in the detection and measurement of fructosamines in a liquid test sample by utilizing a dry phase test strip comprising a test pad including a carrier matrix incorporating an indicator reagent composition. By using the method and device of the present invention, fructosamine levels from about 0.5 mMol/L to about 6 mMol/L, and especially from about 1 mMol/L to about 4 mMol/L, can be determined accurately. Hence, in accordance with the method of the present invention, new and unexpected results are achieved in the dry phase reagent strip assay of blood and other liquid test samples for fructosamines, by utilizing an indicator reagent composition incorporated into a suitable carrier matrix.